Project Summary Small interfering RNAs (siRNAs) are informational drugs that can be designed to treat genetically defined disorders and thereby reshape our approach to human medicine. The clinical utility of siRNAs depends on functional delivery to a tissue and cell type of interest, which is in turn defined by oligonucleotide chemistry. When a chemical architecture?i.e., oligonucleotide modification pattern?that provides functional and non- toxic delivery to a tissue is optimized, candidate drugs can be quickly developed to treat other diseases with the same tissue involvement. Currently, the clinical utility of siRNA is limited to liver, where conjugation of trivalent N-acetylgalactosamine (GalNAc) moiety enables efficient delivery to hepatocytes and therapeutic activity for a year after a single injection. To expand the utility of siRNAs to tissues beyond liver, we must (i) optimize chemical modification patterns that fully stabilize siRNAs and are non-toxic and compatible with the silencing machinery; (ii) understand the mechanisms that define siRNA pharmacokinetic and pharmacodynamic behavior; and (iii) identify and engineer novel ligands that enable targeted tissue delivery and sustained in vivo efficacy. We have the demonstrated expertise in organic chemistry, combinatorial chemistry, oligonucleotide chemistry, RISC biology, and siRNA pharmacology needed to solve these problems. To date, we have identified fully chemically stabilized siRNA scaffolds that exhibit minimal toxicity and immunogenicity; engineered novel conjugates that support functional delivery to liver, kidneys, heart, fat, muscle, and lung; defined chemical approaches to dynamically modulate siRNA clearance; and synthesized novel backbone modifications (phosphonate variants) that improve siRNA stability and, when placed in defined positions, enhance RISC efficacy and specificity. Building on these recent advances, we propose four principal research directions that seek to (i) chemically engineer siRNA scaffolds that enable complete stability and sustained efficacy of any RNA sequence in vivo; (ii) establish phosphonate variants as a new backbone for the modulation of therapeutic RNA properties; (iii) engineer and discover novel ligands that deliver siRNAs to tissues other than liver; and (iv) work with a network of expert collaborators to investigate the therapeutic potential of novel chemical configurations in models of diseases with unmet medical needs. The completion of these studies will establish siRNA chemical architectures that enable functional extrahepatic delivery of siRNAs and lead to the discovery of several compounds with the potential to transform therapeutic approaches for range of diseases.